Ion channels are present in every cell, playing key roles in a range of physiological processes, including cardiac and neural activity. Ion channel disorders have been implicated in epilepsy, cystic fibrosis, malaria, and a number of other diseases (Nilius, B. Biochimica Et Biophysica Acta-Molecular Basis of Disease 1772, 805-812 (2007). Because of their central importance, ion channels are intensely studied scientifically and are critical targets for drugs (Molokanova, M. et al. Drug Discovery Today 13, 14-22 (2008)) as well as the subject of safety concerns for off-target drug interactions (e.g., hERG cardiac K+ channels) (Keating, M. T. et al. Cell 104, 569-580 (2001)).
Electrophysiological measurements of ion channels are complicated by the requirement that they must be incorporated into a lipid bilayer membrane to pass ionic current. These currents can be measured using the techniques of Patch Clamp (primarily used to measure ion channels in cells) or artificial lipid bilayers. Manual patch clamp is regarded as the gold standard for in vitro measurement of ion channels (Dunlop, J., et al., Nature Reviews Drug Discovery 7, 358-368 (2008); Hertzberg, R. P. et al. Current Opinion in Chemical Biology 4, 445-451 (2000), but despite its high quality data, patch clamp's low throughput and high equipment and skill requirements have limited its use to specialists. Ion channel drug screening in industry uses automated patch clamp (APC), an arrayed and automated version of manual patch clamp, which has increased measurement throughput, but is characterized by limited cell compatibility and very high instrumentation and consumable costs, also strongly limiting its use (Comley, J., Drug Discovery World, 47-57 (2003).
In vitro measurement of ion channels in artificial lipid bilayers is well-established for their isolation and study at the single molecule level (Wong, D., Nanotechnology 17, 3710-3717 (2006)) and uses electrical apparatus highly similar to patch clamp (Miller, C. Ion channel reconstitution. (Plenum Press, New York; 1986); Sakmann, B. & Neher, E. (eds.) Single-channel recording. (Plenum Press, New York; 1995)). Artificial bilayers are formed from constituent lipids and will reconstitute ion channels following addition of soluble channels or channel-containing vesicles to the surrounding membrane solution (Miller, C. Ion channel reconstitution. (Plenum Press, New York; 1986)). Ion channel measurement with cell-free artificial bilayers has a number of advantages over patch clamp including reduced equipment and training required and the ability to easily control the membrane composition and surrounding solution. Unfortunately, like patch clamp, it is a manual, low throughput measurement platform suited for specialists.
Electrophysiological activity of ion channels can be measured directly using cell-based patch clamp and cell-free artificial lipid bilayers. However, it is well recognized that these labor intensive platforms also require considerable technical expertise, severely limiting the potential user population as well as the scope and type of measurements that can be conducted. Studies of ion channels and transmembrane proteins in planar lipid bilayer membranes allow for functionality testing in highly controlled environments. Applications ranging from drug interaction testing to mutational studies have been demonstrated. Fully automatable formation and measurement of functional planar lipid bilayers have been shown using the contacting monolayer technique; automated formation of such ‘droplet’ lipid bilayers having consistent and repeatable sizes, however, has not been demonstrated. Further, the ability to perfuse such bilayers during measurement has not been shown.
Reconstitution of ion channels into artificial lipid bilayer membranes enables the isolation and study of individual channels as well as a high degree of control over the membrane composition and surrounding solution. Formation of artificial lipid bilayers from the contact of lipid monolayers self-assembled on oil/aqueous interfaces (Tsofina L M 1966) has been implemented in microfluidic devices (Funakoshi K 2006; Malmstadt 2006) and discrete droplet systems, (Holden M A 2007; Bayley H 2008; Poulos J L 2009) and has been the subject of much recent activity due to its compatibility with automated and parallel implementations (Poulos J L 2009; Poulos J L 2010; Thapliyal 2010) and capability to measure ion channels incorporated directly from primary cells or organelles (Leptihn S 2011).
It was previously shown that bilayer areas are highly sensitive to variations in positioning of the two aqueous phases (Heron A J 2007; Poulos J L 2010), which can in turn affect number of incorporated channels (Leptihn S 2011) and measurement noise (Wonderlin W F 1990; Mayer M 2003).
Thus, there exists a need for devices and methods for producing and measuring artificial bilayers. Such devices and methods are described herein.